REVIEW ARTICLE
Biogenesis of peroxisomes
Topogenesis of the peroxisomal membrane and matrix proteins
Ines Heiland and Ralf Erdmann
Ruhr-Universita
¨
t Bochum, Institut fu
¨
r Physiologische Chemie, Bochum, Germany
Introduction
Peroxisomes are ubiquitious, single membrane bound
organelles of eukaryotic cells [2]. They maintain various
functions that differ depending on the species and cell
type, as well as the environmental or developmental
conditions. Many metabolic pathways of peroxisomes
lead to the production of hydrogen peroxide. The
subsequent decomposition of this toxic compound by
catalase is a fundamental process that takes place in
almost all peroxisomes. Moreover, peroxisomes contrib-
ute to the b- and a-oxidation of fatty acids, synthesis of
ether lipids such as plasmalogens, and the oxidation of
bile acids and cholesterol [3–6]. Defects in the biogenesis
of peroxisomes are the molecular cause for severe inher-
ited diseases, called peroxisome biogenesis disorders
Keywords
peroxin, peroxisome, protein transport
Correspondence
R. Erdmann, Ruhr-Universita
¨
t Bochum,
Institut fu

enigmatic. Recent observations suggest the existence of two classes of per-
oxisomal membrane proteins. Newly synthesized class I proteins are
directly targeted to and inserted into the peroxisomal membrane, while
class II proteins reach their final destination via the endoplasmic reticulum
or a subcompartment thereof, which would be in accord with the idea
that the peroxisomal membrane might be derived from the endoplasmic
reticulum.
Abbreviations
APX, ascorbate peroxidase; mPTS, membrane protein targeting signals; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting
signal; TPR, tetratricopeptide repeat.
2362 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS
(PBD,) such as Zellweger syndrome, neonatal adreno-
leukodystrophy and Refsums disease [7].
Peroxisomal matrix protein import
Many investigations have focussed on the elucidation
of the import of peroxisomal matrix proteins, and the
mechanisms involved are becoming better understood
[8,9]. It is generally accepted that Pex5p and Pex7p,
the receptors for the proteins harboring peroxisomal
targeting sequences, cycle between the cytosol and the
peroxisome. This gave rise to the so-called model of
shuttling receptors [10,11]. According to this model,
the import receptors bind cargo proteins in the cytosol
and direct them to a docking and translocation com-
plex at the peroxisomal membrane. There, the cargo is
released and translocated across the peroxisomal mem-
brane while the receptor shuttles back to the cytosol in
a so-far unknown manner. The so-called extended
shuttle hypothesis is based on the assumption that the
import receptor does not stop at the peroxisomal

However, it has been shown that these proteins are
still targeted via the PTS1 receptor Pex5p, but bind to
regions of the protein distinct from the PTS1-recogni-
tion domain [25]. Pex7p is the cytosolic receptor for
PTS2 proteins and belongs to the family of WD40 pro-
teins that share a consensus sequence of 40 amino
acids, which contains a central tryptophan-aspartic
acid motif [10]. Pex7p contains six of these repeats. In
S. cerevisiae, Pex7p is associated with Pex18p ⁄ Pex21p
[26,27], proteins with redundant functions that are pre-
sumed to mediate the association of cargo-loaded
Pex7p with the docking complex. Whereas Pex7p is
present in nearly all species analysed, Pex18p
and Pex21p are evolutionarily less conserved. In
Neurospora crassa and Yarrowia lipolytica the function
of Pex18p ⁄ Pex21p is performed by Pex20p, suggesting
that the protein is a true orthologue of the yeast pro-
teins [28,29].
In addition to the fact that PTS1 and PTS2 protein
import pathways employ different components there
seems to be a common mechanism for both processes.
In support of this assumption, it has been shown that
Pex18p can functionally replace the N-terminal domain
of Pex5p [30]. Remarkably, in humans, Pex5p exists in
two isoforms, one characterized by a 37 amino acid
insertion that mediates binding of Pex7p to Pex5p and
therefore overcoming the requirement for Pex18p ⁄
Pex21p [31,32]. Thus, in mammalian cells, the PTS2
pathway depends on the presence of the long isoform
of PTS1 receptor Pex5p, which is required to direct

nable that these targeting signals are bound by the
import receptors after cargo release to prevent reas-
sociation with cargo proteins and evidence has been
provided for Pex8p being directly involved in cargo–
receptor dissociation [56]. The functions of other
components of the import complex are still
unknown. Whether the RING-finger complex is
really involved in peroxisomal matrix protein import
or rather in the re-export of the PTS1 receptor
Pex5p still has to be investigated.
It has been demonstrated that Pex5p becomes ubi-
quitinated during import [57–59]. Furthermore,
Pex18p, a component of the signal recognition com-
plex in the PTS2-pathway, becomes mono- and diubiq-
uitinated during import and is degraded in a
proteasome-dependent manner [60]. Polyubiquitination
of Pex5p is detectable in pex1, pex6, pex4 and pex22
mutants of S. cerevisiae and requires a functional
import complex. The physiological relevance of Pex5p
ubiquitination, however, remains to be shown. It is
possible that import receptors that remained in the
import pathway are polyubiquitinated and subse-
quently directed to proteasomal degradation as a form
of quality control [58]. However, it is also conceivable
that ubiquitination of Pex5p and Pex18p serves as a
signal for their export back to the cytosol [57,59]. As
RING-finger proteins often function as E3–ubiquitin
protein ligases in ubiquitin and ubiquitin-like conjuga-
tions [61], Pex2p, Pex10p and Pex12p might be
involved in the ubiquitination of the import receptor.

icles as postulated by Purdue and Lazarow [68]. As an
alternative, membrane constituents might flip from the
ER membrane at contact sites between ER and peroxi-
somes. Evidence has been provided that the latter
mechanism is employed for the transport of phospho-
lipids from the ER to mitochdondria [69–71]. How
peroxisomes gain their phospholipids remains to be
investigated.
Peroxisomal membrane protein import
Most mutants that are defective for the import of
PTS1 and PTS2 proteins still import peroxisomal
membrane proteins. Thus, the import of peroxisomal
membrane and matrix proteins is independent
[41,42,72]. The peroxisomal membrane protein target-
ing signals (mPTS) were identified for several peroxi-
somal membrane proteins (PMPs). These targeting
sequences contained a basic amino acid sequence in
conjunction with at least one transmembrane region
[73–77].
Some PMPs have been shown to posses multiple tar-
geting signals [55,78,79]. One possible reason for the
existence of multiple mPTS might be that they are
required to distinguish targeting to different peroxi-
some populations [55]. This might be of particular
interest for higher eukaryotes such as plants, which
generate different types of peroxisomes during their
development.
Only three of the 32 peroxins identified so far –
Pex3p, Pex16p and Pex19p – have been shown to be
involved in peroxisomal membrane protein import

by the Pex19p binding site together with an adjacent
transmembrane segment. In this assembly, the Pex19p
binding site is proposed to contain the required
targeting information, while the transmembrane seg-
ment is required for the permanent insertion of the
protein into the peroxisomal membrane. The fact that
the Pex19p binding site is an integral part of the mPTS
also demonstrates that Pex19p functions as a targeting
sequence receptor for peroxisomal membrane proteins.
There is, however, one exception. Pex3p targeting is
not dependent on Pex19p, and Pex19p binds to Pex3p
in regions different from its targeting signal [90,92].
Therefore, the existence of distinct classes of peroxi-
somal membrane proteins have been postulated
[93,94]. Class I PMPs are synthesized on free ribo-
somes in the cytosol and require Pex19p for their post-
translational import into the peroxisome. Class II
PMPs, such as Pex3p, are targeted to the peroxisome
independent of Pex19p [92].
The function of Pex19p as an mPTS receptor does
not exclude that binding could contribute to the stabil-
ity of the proteins [95]. In fact, Pex19p has been shown
to increase the half-life of newly synthesized membrane
proteins in vivo [78], and it has been demonstrated to
bind to in vitro synthesized Pmp22p and thereby main-
tain its solubility [92]. This could be explained by
mPTS itself being rather hydrophobic, and thus, if not
shielded from hydrophobic environment, it might
Fig. 1. PTS1-import model. Newly synthes-
ized peroxisomal matrix proteins are recog-

targeting signal, whereas its C-terminus binds Pex19p
at regions distinct from the PMP binding site. The
interaction of Pex19p with Pex3p is essential for peroxi-
somal membrane protein import, suggesting that Pex3p
functions as a receptor for Pex19p at the peroxisomal
membrane [92,93]. It is now thought that Pex19p recog-
nizes newly synthesized PMPs in the cytosol and directs
them to the peroxisomal membrane, probably via bind-
ing to Pex3p. How peroxisomal membrane proteins
insert into the membrane remains to be investigated.
As outlined above, the topogenesis of Pex3p seems
to be different from that of other PMPs. The N-ter-
minal 50 amino acids of Pex3p have been shown to be
associated with vesicles that are located close to the
nucleus in Dpex3 mutants of H. polymorpha. Further-
more, these vesicles are reported to be capable of
forming mature peroxisomes after complementation
with full length Pex3p [97]. The first 16 amino acid of
Pex3p lead to targeting of reporter constructs to the
ER [98]. Whether this targeting sequence is functional
in the endogenous Pex3p is not known.
Involvement of the endoplasmic
reticulum in peroxisome biogenesis
In early years, it was assumed that peroxisomes origin-
ate through budding from the endoplasmic reticulum
[99]. In 1984, however, Fujiki and coworkers demon-
strated that the peroxisomal membrane protein
Pmp22p is synthesized on free ribosomes in the cytosol
and imported post-translationally directly into peroxi-
somes [100]. Based on these and other data, the

Kar2p and a cytosolic mislocalization of thiolase and
alcohol oxidase in secretory pathway mutants (sec-
mutants) of Yarrowia lipolytica [107]. Furthermore,
evidence for involvement of the ER in peroxisome bio-
genesis was provided by Mullen and coworkers, who
demonstrated that tail-anchored peroxisomal mem-
brane proteins such as APX and Pex15p are imported
into plant microsomes in vitro, whereas Pmp45p is
imported directly into peroxisomes [102,108]. Further-
more, Tabak and coworkers reported on reticular
structures observed in untreated mouse dendritic cells
that contained PMPs and were connected to the
smooth ER [109,110].
Taken together, there is striking evidence for an
involvement of the ER in peroxisome biogenesis. How-
ever, the data are clear in that the standard secretion
pathway is not involved. The only way to reconcile
these facts seems to propose the existence of a new
route for the insertion of peroxisomal proteins into the
ER membrane. In this respect, it is interesting to note
that several new routes for protein transport into the
ER have been identified in recent years that do not or
only partially employ the standard secretion pathway.
One of these novel import pathways into the ER is the
topogenesis of Ist2p. The import of Ist2p is mRNA-
dependent and takes place at the cortical ER of the
daughter cell [111]. Whether this process requires
Sec61p is unknown. An example of sec-independent
import into the ER is the sorting of Nyv1p. This
tail-anchored protein has been shown to be imported

anchored peroxisomal membrane proteins (e.g. Pex15p
and APX) that are supposed to be targeted to a thus
far uncharacterized circular reticular membrane com-
partment, namely peroxisomal ER or peroxisomal
reticulum. These reticular structures may, at least
temporally, be connected to the ER or may even repre-
sent an ER subdomain [110]. Consequently, newly syn-
thesized proteins of class II might first be inserted into
the ER membrane before they reach their final destina-
tion in the peroxisomal membrane in an unknown
fashion. Nevertheless, in the presence of mature per-
oxisomes these proteins might also behave like PMPs
of type I and thus be imported preferentially directly
into peroxisomes. In the absence or deficiency of per-
oxisomal membranes, these proteins might be imported
into the reticular structures and contribute to the
de novo synthesis of peroxisomes. Whether the topo-
genesis pathway of these PMPs shares components
with other sec-independent transport pathways remains
to be investigated.
Acknowledgements
We thank Hanspeter Rottensteiner and Wolfgang
Schliebs for reading the manuscript. Ines Heiland was
supported by a Boehringer Ingelheim Fonds fellow-
Fig. 2. Model of peroxisomal membrane
biogenesis. Peroxisomal class I membrane
proteins are synthesized on free ribosomes
in the cytosol, where they are recognized by
the import receptor Pex19p that directs
them to the peroxisomal membrane. Mem-

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